The goal of this project is to understand the molecular mechanisms of early stages of replication of a virus with an RNA genome. Viruses with similar RNA genomes include potent human and animal pathogens, including Ebola, measles, and vesicular stomatitis virus (VSV). VSV is the model being studied in this project. To read and copy their RNA genomes, VSV and related RNA viruses make a special RNA polymerase that makes RNA copies of the viral RNA genomes, and therefore are called "RNA dependent RNA Polymerases" or RdRPs. This project will examine the mechanism by which RdRPs read and copy the viral genome template in infected cells which is critical for the replication of viruses. The results from this study will improve scientific understanding of how RdRPs work and identify potential target mechanisms for inhibition of viral infections. In addition to furthering knowledge about RNA virus replication, this study will train minority undergraduate students by incorporating them within teams doing research at the University of Utah and UMass Medical School at Worcester. The proposed activities will also support development of Biophysics courses that will benefit undergraduate interdisciplinary education and encourage participation of K-12 children in science by communicating aspects of the scientific results through creation of virus dance projects for children.
This project will use single molecule live cell imaging to visualize transcription events from single VSV genome templates in infected cells and utilize fluorescence correlation spectroscopy to measure the concentration of free RdRPs during transcription in vivo. When stretched, the genome template of VSV is more than 4 microns in size and RdRPs can only initiate transcription on its 3' end. While 50 RdRPs are tightly bound to this template, how they initiate and sustain transcription is not clear. The live cell imaging experiments will determine the number of active RdRPs per genome and their cooperativity. Measurements of the concentration of free RdRPs not bound to the genome will verify if a dissociation and 3' binding mechanism can support viral transcription in vivo. This project will also use reconstituted templates in vitro to visualize the mechanism by which RdRPs redistribute on the genome template. We have observed single RdRP sliding on purified genome templates in vitro. In this project we will further characterize the sliding mechanism of RdRPs on genome templates. By measuring the dynamics of RdRPs in vivo as well as in vitro this project will determine the mechanism of transcription by RdRPs during VSV RNA virus infection.
The project was funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.
